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Characterization of four BCHE mutations associated with prolonged effect of suxamethonium

The Pharmacogenomics Journal volume 21, pages 165–173 (2021)Cite this article

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Abstract

Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a “silent” BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower kcat value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (Ω-loop) can explain the modification of the kinetic parameter of the mutated protein.

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Fig. 1: Spatial distribution of the different BChE mutations reported in this study.
Fig. 2: Electrophoretic analyses of plasma of patients with BChE activity staining or immunostaining.
Fig. 3: Enzymatic activity of the different recombinant BChE constructs transiently expressed in CHO cells.
Fig. 4: Distance evolution between serine histidine active site residues during molecular dynamic simulations.
Fig. 5: Evolution of the gyration radii of wild-type and ∆21, ∆I140, R386C and L88H mutants of BChE over 100-ns-long molecular dynamic simulations.
Fig. 6: b-factors of backbone atoms of wild-type and L88H-BChE calculated over 100-ns-long molecular dynamic simulations.

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References

  1. Lockridge O. Review of human butyrylcholinesterase structure, function,genetic variants, history of use in the clinic, and potential therapeutic uses. Pharmacol Ther. 2015;148:34–46.

    Article  CAS  Google Scholar 

  2. Schopfer LM, Lockridge O, Brimijoin S. Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin. Gen Comp Endocrinol. 2015;224:61–8.

    Article  CAS  Google Scholar 

  3. Chen VP, Gao Y, Geng L, Parks RJ, Pang YP, Brimijoin S. Plasma butyrylcholinesterase regulates ghrelin to control aggression. Proc Natl Acad Sci USA. 2015;112:2251–6.

    Article  CAS  Google Scholar 

  4. Brimijoin S, Chen VP, Pang YP, Geng L, Gao Y. Physiological roles for butyrylcholinesterase: a BChE-ghrelin axis. Chem Biol Interact. 2016;259:271–5.

    Article  CAS  Google Scholar 

  5. Forbat A, Lehmann H, Silk E. Prolonged apnoea following injection of succinylcholine. Lancet. 1953;265:1067–8.

    CAS  PubMed  Google Scholar 

  6. Kalow W, Genest K. A method for the detection of atypical forms of human serum cholinesterase; determination of dicucaine number. Can J Biochem Physiol. 1957;35:339–46.

    Article  CAS  Google Scholar 

  7. Delacour H, Ceppa F. Déficit en butyrylcholinestérase: quelle démarche diagnostique, quelles conséquences pour le patient? Anest Reanim. 2017;3:99–100.

    Google Scholar 

  8. Masson P, Legrand P, Bartels CF, Froment MT, Schopfer LM, Lockridge O. Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase. Biochemistry. 1997;36:2266–77.

    Article  CAS  Google Scholar 

  9. Podoly E, Shalev DE, Shenhar-Tsarfaty S, Bennett ER, Ben Assayag E, Wilgus H, et al. The butyrylcholinesterase K variant confers structurally derived risks for Alzheimer pathology. J Biol Chem. 2009;284:17170–9.

    Article  CAS  Google Scholar 

  10. David SM, Venkatesan SK, Boopathy R. An Indian butyrylcholinesterase variant L307P is not structurally stable: a molecular dynamics simulation study. Chem Biol Interact. 2013;203:30–5.

    Article  CAS  Google Scholar 

  11. Delacour H, Lushchekina S, Mabboux I, Ceppa F, Masson P, Schopfer LM, et al. Characterization of a novel butyrylcholinesterase point mutation (p.Ala34Val), “silent” with mivacurium. Biochem Pharmacol. 2014;92:476–83.

    Article  CAS  Google Scholar 

  12. Delacour H, Lushchekina S, Mabboux I, Bousquet A, Ceppa F, Schopfer LM, et al. Characterization of a novel BCHE “silent” allele: pointmutation (p.Val204Asp) causes loss of activity and prolonged apnea with suxamethonium. PLoS ONE. 2014;9:e101552.

    Article  Google Scholar 

  13. Lushchekina S, Delacour H, Lockridge O, Masson P. Human butyrylcholinesterase polymorphism: Molecular modeling. Int J Risk Saf Med. 2015;27:S80–1.

    Article  Google Scholar 

  14. Radic Z, Pickering NA, Vellom DC, Camp S, Taylor P. Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry. 1993;32:12074–84.

    Article  CAS  Google Scholar 

  15. Nachon F, Nicolet Y, Viguie N, Masson P, Fontecilla-Camps JC, Lockridge O. Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization. Eur J Biochem. 2002;269:630–7.

    Article  CAS  Google Scholar 

  16. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.

    Article  CAS  Google Scholar 

  17. Choi Y, Chan AP. PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics. 2015;31:2745–47.

    Article  CAS  Google Scholar 

  18. Karnovsky MJ, Roots LA. “Direct-coloring” thiocholine method for cholinesterases. J Histochem Cytochem. 1964;12:219–21.

    Article  CAS  Google Scholar 

  19. Chiu J, March PE, Lee R, Tillett D. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Res. 2004;32:e174.

    Article  Google Scholar 

  20. Lockridge O, Blong RM, Masson P, Froment MT, Millard CB, Broomfield CA. A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase. Biochemistry. 1997;36:786–95.

    Article  CAS  Google Scholar 

  21. Brazzolotto X, Wandhammer M, Ronco C, Trovaslet M, Jean L, Lockridge O, et al. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure. FEBS J. 2012;279:2905–16.

    Article  CAS  Google Scholar 

  22. Webb B, Sali A. Protein structure modeling with MODELLER. Methods Mol Biol. 2014;1137:1–15.

    Article  CAS  Google Scholar 

  23. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25.

    Article  Google Scholar 

  24. Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins. 2006;65:712–25.

    Article  CAS  Google Scholar 

  25. Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps JC, Nachon F. Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products. J Biol Chem. 2003;278:41141–7.

    Article  CAS  Google Scholar 

  26. Leung MR, van Bezouwen LS, Schopfer LM, Sussman JL, Silman I, Lockridge O, et al. Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly. Proc Natl Acad Sci USA. 2018;115:13270–5.

    Article  CAS  Google Scholar 

  27. Masson P, Carletti E, Nachon F. Structure, activities and biomedical applications of human butyrylcholinesterase. Protein Pept Lett. 2009;16:1215–24.

    Article  CAS  Google Scholar 

  28. Yen T, Nightingale BN, Burns JC, Sullivan DR, Stewart PM. Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population. Clin Chem. 2003;49:1297–308.

    Article  CAS  Google Scholar 

  29. Karczewski K, Francioli L, Tiao G, Cummings B. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. 2019. https://www.biorxiv.org/content/10.1101/531210v4.

  30. Masson P, Froment MT, Bartels CF, Lockridge O. Asp70 in the peripheral anionic site of human butyrylcholinesterase. Eur J Biochem. 1996;235:36–48.

    Article  CAS  Google Scholar 

  31. De Jaco A, Comoletti D, Kovarik Z, Gaietta G, Radic Z, Lockridge O, et al. A mutation linked with autism reveals a common mechanism of endoplasmic reticulum retention for the alpha,beta-hydrolase fold protein family. J Biol Chem. 2006;281:9667–76.

    Article  Google Scholar 

  32. Zhang X, Lin H, Zhao H, Hao Y, Mort M, Cooper DN, et al. Impact of human pathogenic micro-insertions and micro-deletions on post-transcriptional regulation. Hum Mol Genet. 2014;23:3024–34.

    Article  CAS  Google Scholar 

  33. The Uniprot Consortium. Uniprot: a worldwild hub of protein knowledge. Nucleic Acid Res. 2019;47:D506–15.

  34. Mikami LR, Wieseler S, Souza RL, Schopfer LM, Lockridge O, Chautard-Freire-Maia EA. Expression of three naturally occurring genetic variants (G75R, E90D, I99M) of the BCHE gene of human butyrylcholinesterase. Pharmacogenet Genom. 2007;17:681–5.

    Article  CAS  Google Scholar 

  35. Schrodinger LLC. The PyMOL molecular graphics system. Version. 2010;1:3r1.

    Google Scholar 

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Acknowledgements

XB, VG, AI and FN were supported by the French Ministry of Armed Forces (Direction Générale de l’Armement et Service de Santé des Armées) under project NBC-5-C-4210 and PDH-2-NRBC-3-C-3201.The authors would like to thank Prof. Oksana Lockrige and Dr. Eric Krejci for their technical advices.

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Authors and Affiliations

  1. Institut de Recherche Biomédicale des Armées, Division Défense NRBC, Département de Toxicologie et Risques Chimiques, 1 place Général Valérie André, B.P. 73, 91223, Brétigny sur Orge Cedex, France

    Xavier Brazzolotto, Virginia Guillon, Alexandre Igert, Joseph Kononchik & Florian Nachon

  2. Hôpital d’Instruction des Armées Bégin, Fédération des Laboratoires, 69 avenue de Paris, 94163, Saint-Mandé Cedex, France

    Sébastien Courcelle, Christophe Sauvanet, Franck Ceppa & Hervé Delacour

  3. Ecole du Val-de-Grâce, 1 place Alphonse Laveran, 75230, Paris Cedex 05, France

    Franck Ceppa & Hervé Delacour

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  1. Xavier Brazzolotto
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  2. Sébastien Courcelle
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  3. Christophe Sauvanet
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  4. Virginia Guillon
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  5. Alexandre Igert
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  9. Hervé Delacour
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Contributions

Concept and design: XB, HD. Data collection: SC, CS, VG, AI. Data analysis: XB, FN, HD. Writing: XB, HD, JK. Critical review: FN, FC.

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Correspondence to Hervé Delacour.

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Brazzolotto, X., Courcelle, S., Sauvanet, C. et al. Characterization of four BCHE mutations associated with prolonged effect of suxamethonium. Pharmacogenomics J 21, 165–173 (2021). https://doi.org/10.1038/s41397-020-00192-7

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